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Technical Briefs |
1 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT
2 ARUP Laboratories, Inc., Salt Lake City, UT
3 Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT
aaddress correspondence to this author at: Department of Pathology, University of Utah, c/o ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108; fax 801-584-5207, e-mail e.frank{at}aruplab.com
Measurement of plasma metanephrine and normetanephrine concentrations has recently been acknowledged as one of the leading tests for the diagnosis of the neuroendocrine tumor, pheochromocytoma (1). The occurrence of the tumor is rare, and it is often difficult to diagnose. The wide spectrum of signs and symptoms includes hypertension, headaches, and diaphoresis. Located primarily in the adrenal gland, these tumors produce excess catecholamines and their metabolites. Assays have been developed to measure catecholamines and metanephrines in both plasma and urine. Although tests for all of these compounds can be diagnostically useful, measurement of metanephrine and normetanephrine in plasma has been reported as a particularly sensitive and specific indicator of the disease. The utility of the test is limited by uncertainty of the predictive value of measured concentrations. This is attributable in part to ambiguity in published reference intervals for these analytes. Lenders et al. (1) reported upper reference limits of 0.3 nmol/L for plasma metanephrine and 0.6 nmol/L for normetanephrine, whereas a reference laboratory (2) reported values <0.5 nmol/L for metanephrine and <0.9 nmol/L for normetanephrine as within the reference interval.
In this study, we established a reference interval by measuring metanephrine and normetanephrine concentrations in plasma collected from 120 volunteers after obtaining informed consent. Reference individuals (53 females and 67 males; age range, 20–73 years) fasted for 12 h before sampling. Interviews were conducted to excuse individuals taking medications, particularly for hypertension. All participants were sitting upright as their blood was collected by needlestick. The whole blood, in EDTA, was stored at 2–8 °C until it was centrifuged within a few hours after collection. Plasma was separated and stored at –70 °C until analysis.
The conditions under which the volunteers were prepared and blood was collected were selected based on evaluation of studies in the literature. Volunteers were fasting to eliminate the interference of dietary constituents in the HPLC analysis, as observed by Lenders et al. (1). For this reason, medical practitioners are advised to instruct patients to fast for 12 h before blood collection for plasma metanephrine analysis. Additionally, as published elsewhere, plasma catecholamine concentrations are posture-dependent, but plasma metanephrine concentrations are not significantly altered if a patient is sitting or lying supine (3).
Metanephrines were assayed by a procedure similar to the method developed by Lenders et al. (4). Contaminants were removed by solid-phase extraction with BondElut® AccuCAT columns (Varian, Inc.). Plasma (1 mL) was applied to the columns with an internal standard, 4-hydroxy-3-methoxybenzylamine. The analytes were eluted with 10% ammonium hydroxide in methanol (1:3 by volume), and the eluate was evaporated to dryness under nitrogen at 37 °C. The residue was reconstituted in 115 µL of 0.2 mol/L acetic acid, and 80 µL was injected for separation by reversed-phase HPLC. The mobile phase consisted of acetonitrile (70 mL/L) in sodium phosphate buffer, pH 3.25. The mobile phase flow rate was 1.2 mL/min. The analytical column was a LUNA C18 reversed-phase column [250 x 4.6 mm (i.d.); 5-µm bead size; Phenomenex]. The ESA 580 pump, 540 autosampler, and ESA 316 CoulArray Detector were from ESA, Inc. The analytes were detected coulometrically with a conditioning cell (5021; ESA) electrode at 410 mV and an analytical cell (5011; ESA) at –310 mV. All of the compounds were separated and detected within 20 min. The retention times for metanephrine, normetanephrine, and the internal standard were 9.5, 7.1, and 11.7 min, respectively. The total run-time was extended to 31 min because of late-eluting compounds. A five-point calibration curve with the internal standard was used to calibrate the method. Analytes were quantified by comparison of the peak height and internal standard ratio with the calibration curve.
A range of concentrations of metanephrine and normetanephrine was injected to determine the linearity of the method. The recovery of this method was established by analyzing solutions of known concentrations and plotting the known samples against the values acquired by the analysis. Over the linear range of the method, recovery for metanephrine was 100.3% of the expected amount and recovery for normetanephrine was 102.5%. Imprecision was determined by assaying each of three quality-control solutions in duplicate on 15 different days. This method was compared with a similar HPLC method (with cation-exchange, solid-phase extraction, and electrochemical detection) at a reference laboratory. Thirty samples, with concentrations spanning the linear range of the method, were analyzed by both methods, and data were evaluated by plotting the points for comparison. For metanephrine, the standard error of the estimate (Sy|x) was 0.20 nmol/L, unfortunately high because of a difference in detection limits for the two methods. Results >0.15 nmol/L were calculated by this method, but the reference laboratory did not quantify results <0.2 nmol/L. The standard error of the estimate was 0.28 nmol/L for normetanephrine. The performance characteristics data are summarized in Table 1
. Acetaminophen, caffeic acid, and ephedrine did not interfere with this assay at physiologic concentrations.
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The reference interval data were analyzed according to the NCCLS guideline for determining reference values (5). Data points were placed in decreasing order and then evaluated to eliminate outliers. The distribution of results was nongaussian for both analytes (Fig. 1
). The central 95% of the data were taken as the reference interval for each analyte.
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Results ranged from <0.15 to 0.37 nmol/L for metanephrine and from <0.16 to 0.94 nmol/L for normetanephrine. Fifty-three percent of the metanephrine data fell below 0.15 nmol/L, the limit of quantification for this assay. The mean and median values were 0.16 and 0.15 nmol/L, respectively, for metanephrine and 0.40 and 0.36 nmol/L for normetanephrine. With 90% confidence, the upper reference limits were determined to be 0.29 nmol/L for metanephrine and 0.77 nmol/L for normetanephrine. The reference intervals calculated in this study were comparable to those reported previously in the literature and fall between those of the two cited reports (1)(2). Reference interval values are included in Table 1
.
We analyzed patient results over a 6-month period with the newly determined reference limits. Of 1657 patient samples, 20.0% were increased with our upper reference limit, 0.29 nmol/L; 17.6% of the metanephrine values were increased by the criterion of 0.5 nmol/L suggested by Sawka et al. (2). For normetanephrine, 25.1% were increased based on our upper reference limit of 0.77 nmol/L, and 4.6% of the values were increased according to the upper reference limit of 0.9 nmol/L reported by Sawka et al. (2).
As reported by Kudva et al. (6), cutoff concentrations for some analytes used to diagnose pheochromocytoma are adjusted to approximately twice the upper limit found in a healthy population. This technique has been found to provide optimal specificity while maintaining acceptable sensitivity. Although the authors do not state that this method was used to establish reference limits for plasma metanephrine and normetanephrine concentrations, it is interesting to note that this would account for the differences between reference limits used by Sawka et al. (2) and those determined in our study of a healthy population as well as those used by Lenders et al. (1). Definition of a positive result as a multiple of the upper limit of the 95% population reference interval may be of value in minimizing false-positive results and is consistent with the accepted medical use of biochemical testing to aid clinical judgment in the evaluation of pheochromocytoma (7). Adoption of lower reference limits for metanephrine and normetanephrine, such as those determined in our study, may lead to an increase in reported positive results. In this case, additional biochemical testing and radiologic imaging will be required for the diagnostic confirmation of pheochromocytoma. Although false-positive tests may lead to increased costs to the patient, false-negative results are far more destructive and are potentially fatal.
This study established concise numerical limits for metanephrine and normetanephrine concentrations in the plasma of a population of healthy individuals with no indication of disease. The results of the study may be of value in the interpretation of plasma metanephrine concentrations used to support a clinical diagnosis of pheochromocytoma.
Acknowledgments
Research for this work was supported by the ARUP Institute for Clinical and Experimental Pathology.
References
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